WO2018006014A1 - Compositions et procédés servant au pronostic d'une infection grippale - Google Patents

Compositions et procédés servant au pronostic d'une infection grippale Download PDF

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WO2018006014A1
WO2018006014A1 PCT/US2017/040368 US2017040368W WO2018006014A1 WO 2018006014 A1 WO2018006014 A1 WO 2018006014A1 US 2017040368 W US2017040368 W US 2017040368W WO 2018006014 A1 WO2018006014 A1 WO 2018006014A1
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pde3a
primer
influenza
patient
infection
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PCT/US2017/040368
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WO2018006014A8 (fr
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Johnson Yiu-Lam LAU
Kai Wang Kelvin TO
Je ZHOU
Johnson Yiu-Nam Lau
Kwok-Yung Yuen
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Emerging Viral Diagnostics (Hk) Limited
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Priority to US16/312,958 priority Critical patent/US11441198B2/en
Priority to CN201780050956.4A priority patent/CN109937260B/zh
Publication of WO2018006014A1 publication Critical patent/WO2018006014A1/fr
Publication of WO2018006014A8 publication Critical patent/WO2018006014A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/30—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for calculating health indices; for individual health risk assessment

Definitions

  • the field of the invention is compositions and methods for providing a prognosis for a viral infection, in particular an influenza infection.
  • One of the antiviral strategies is to target host cell machineries required for viral replication.
  • the potential benefits of using host-directed antivirals include a lower likelihood for viruses to develop resistance, and a broader spectrum of antiviral activity if different viruses rely on the same mechanism for viral replication.
  • host-directed antivirals For influenza virus, several clinically available host-directed drugs have undergone clinical trials.
  • DAS 181 a sialidase which cleaves the sialic acids of the cellular receptor for influenza virus (see United States Patent Application
  • Publication No. 2016/0052905, to Lee et al. has antiviral activity against influenza viruses.
  • the first approach is to identify host factors that are important for viral replication. This can be achieved by using in vitro screening systems, with the importance of each gene verified by knockdown or over-expression of the candidate gene in vitro or in animal models.
  • the second approach is to identify genetic variations that are significantly different between severe and mild cases [14]. Using the latter approach CD55, SFTPB, TMPRSS2 and LGALS 1 have been identified as host susceptibility genes for the 2009 pandemic influenza A(H1N1) (A[HlNl]pdm09) or A(H7N9) virus infection (see To KK, Zhou J, Song YQ, et al.
  • Surfactant protein B gene polymorphism is associated with severe influenza. Chest 2014; 145: 1237-43; Zhou J, To KK, Dong H, et al. A functional variation in CD55 increases the severity of 2009 pandemic H1N1 influenza A virus infection. J Infect Dis 2012; 206:495-503; Chen Y, Zhou J, Cheng Z, et al. Functional variants regulating LGALS 1 (Galectin 1) expression affect human susceptibility to influenza A(H7N9). Sci Rep 2015; 5:8517; Cheng Z, Zhou J, To KK, et al. Identification of TMPRSS2 as a
  • compositions and methods that provide prognostic information to a medical practitioner treating influenza.
  • the inventive subject matter provides apparatus, systems and methods in which the prognosis for an individual that is infected with an influenza virus can be estimated by determining the presence or absence of certain genetic mutations in that individual. Suitable genetic mutations include a PDE3A mutation, where severity of the viral infection is increased when the PDE3A mutation results in reduced PDE3A function.
  • One embodiment of the inventive concept is a method of providing a prognosis for an individual by testing a sample (such as such as blood, saliva, a nasal swab, and/or a cheek swab) obtained from an individual for the presence of a PDE3A mutation associated with reduced PDE3A function.
  • the mutation can be a single nucleotide polymorphism (SNP), for example the one or more of the SNPs rs7314545, rs6487131, and/or rs6487132.
  • SNP single nucleotide polymorphism
  • the presence of such a mutation can be determined by genotyping (for example, by polynucleotide amplification) or by characterization of a gene produce (for example, by an immunochemical assay).
  • the reduction in PDE3A can be due to reduced activity of the gene product, reduced PDE3A gene
  • kits for providing a prognosis for an influenza infection which includes a first primer that is at least partially complementary to a PDE3A gene or gene transcript; and instructions for use.
  • the primer can include a non-naturally occurring base and/or a non-naturally occurring sugar-phosphate backbone analog.
  • the first primer is designed to permit detection of a SNP, such as rs7314545, rs6487131, and rs6487132.
  • first primer includes a polynucleotide sequence that is at least partially complementary to a region of PDE3A from which an intron has been excised.
  • the kit can include an additional, different primer that is at least partially complementary to a PDE3A gene or gene transcript, and that is designed to act in concert with the first primer in a polynucleotide amplification reaction.
  • Such kit can also include a probe that is at least partially complementary to PDE3A or a PDE3A amplification product, wherein the probe comprises a detectable tag.
  • Another embodiment of the inventive concept is a primer for providing a prognosis for influenza infection, which includes a polynucleotide sequence that is at least partially
  • the primer can include a non- naturally occurring base and/or a non-naturally occurring sugar-phosphate backbone analog. Such a primer can be designed to permit detection of a SNP, such as rs7314545, rs6487131, and/or rs6487132.
  • the primer can include a polynucleotide sequence that is at least partially complementary to a region of PDE3A from which an intron has been excised.
  • Another embodiment of the inventive concept is a method for assessing the clinical outcome of an influenza infection, by providing an algorithm that includes a correlation between the presence of at least one PDE3A mutation and a severe outcome of influenza infection and inputting a patient' s PDE3A status to the algorithm.
  • Such an algorithm can be a multivariate analysis that incorporates one or more additional patient factors, such as patient age, patient gender, patient immune status, patient ethnicity, patient immune status, patient lifestyle factors, presence of non-infectious disease, viral genetic factors, and/or the presence of non-PDE3A genetic markers of the patient.
  • Another embodiment of the inventive concept is a method for treating an influenza infection, by increasing the activity of a pro-inflammatory cytokine (such as TNFa, IL- ⁇ ⁇ , IL-6, and/or IL-32) in an infected individual.
  • a pro-inflammatory cytokine such as TNFa, IL- ⁇ ⁇ , IL-6, and/or IL-32
  • the increase in activity can be accomplished by supplying the pro-inflammatory cytokine to the individual as a prodrug and/or prodrug conjugate (e.g. a polyethylene glycol conjugate).
  • the activity is increased by providing a pro-inflammatory cytokine agonist.
  • the activity is increased by increasing expression and/or release of a pro-inflammatory cytokine.
  • FIG. 1 depicts an allelic specific expression profile of PDE3A SNPs in human left ventricle tissue.
  • FIGs. 2A to 2C show differences in genetic risk of development of severe symptoms following viral influenza infection among different populations.
  • FIG. 2A depicts SNP frequency distribution in populations experiencing severe or mild influenza.
  • FIG. 2B shows the proportions of Chinese and Caucasian populations having high, elevated, and typical genetic risks for developing severe illness on infection with HlNl pdm09 influenza virus infection. Populations shown are Southern Chinese in Macau; (Macau), Southern Chinese (CHS), Beijing Chinese (CHB), European (EUR), and American (AMR).
  • FIG. 2C shows a comparison of population genetic risk distributions of Chinese and Caucasian (upper panel), and Chinese populations from different regions (lower panel). Cumulative distributions of genetic risks of having severe illness after H1N1 pdm09 infection are plotted against population percentage. Populations are designated as in FIG. 2B.
  • FIGs. 3A and 3B show the effect of viral infection on PDE3A.
  • FIG. 3A shows increased PDE3A expression following viral infection.
  • FIG. 3B shows increased PDE3A mPvNA expression following viral infection.
  • FIGs. 4A and B show the results of PDE3A siRNA knockdown studies.
  • FIG. 4A depicts the effect of PDE3A siRNA knockdown on PDE3A expression.
  • FIG. 4B depicts the effect of siRNA knockdown on PDE3A protein concentration.
  • FIGs. 5A to 5C show the effect of siRNA on viral replication in treated cells.
  • FIGs. 5A and 5B depict the effect of PDE3A siRNA on viral titer for various influenza strains in transfected cells at 24 hours and 48 hours post-infection, respectively.
  • FIG. 5C shows expression of influenza virus nucleoprotein in cells transfected with PDE3A siRNA.
  • FIGs. 6A and 6B show the results of PDE3B siRNA knockdown studies.
  • FIG. 6A depicts the effect of siRNA knockdown on PDE3B expression.
  • FIG. 6B depicts the effect of PDE3B siRNA on influenza virus titer in transfected cells.
  • FIG. 7A and 7B show the results if siRNA knockdown on cytokine expression in influenza infected cells.
  • FIG. 7A shows typical result for IL-6 expression.
  • FIG. 7B shows typical results for IL-32 expression.
  • the inventive subject matter provides compositions and methods that permit identification of individuals with an increased risk of severe (e.g. life threatening) sequelae following infection with an influenza virus.
  • the inventors have identified a previously unsuspected gene, PDE3A, using a 2-tier genetic association study, and have further identified single nucleotide polymorphisms (SNPs) associated with the development of severe disease, specifically rs7314545, rs6487131, and rs6487132.
  • SNPs single nucleotide polymorphisms
  • Such genetic markers can be used in isolation or in combination with other patient-specific factors (e.g.
  • inventive subject matter provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
  • PDE3A a host factor influencing the disease severity of influenza virus (for example influenza A(HlNl)pdm09) infection based on genetic association studies comparing severe and mild influenza cases, and between severe influenza cases and the general population. Multivariate analysis confirmed that the genetic association between PDE3A SNPs rs7314545/rs6487132 and influenza infection was not due to known clinical risk factors. In addition in vitro studies showed that influenza virus infection induced the expression of PDE3A.
  • Embodiments of the inventive concept include compositions and methods for
  • Methods of the inventive concept can include methodologies that can identify the presence of mutations in the PDE3A gene that negatively affect expression and/or function of the gene product as predisposing an individual having such a mutation to severe complications and/or death on infection with an influenza virus.
  • Such mutations include mutations in upstream sequences that influence transcription, translocations, substitutions of one or more nucleotides (for example, a single nucleotide polymorphisms or SNP), deletions, and insertions of a PDE3A gene, a regulatory element associated with a PDE3A gene, and/or a gene involved in regulation of a PDE3A gene.
  • the mutation is embodied in one or more SNPs within a structural and/or functional portion and/or region of PDE3A and/or an associated regulatory region.
  • Such mutations can be identified using statistical techniques related to sequelae of influenza infection as applied to genomic data, as described below.
  • Identification of the presence of such a PDE3A mutation in an individual can be used prognostically as an indicator of the severity of an influenza infection, and thereby provide guidance to a healthcare provider in determining a course of treatment.
  • other mutations shown to be linked to PDE3A mutations so identified can be utilized to identify an individual susceptible to severe influenza infection. Examples of suitable mutations include the SNPs designated rs7314545, rs6487131, and rs6487132.
  • patient genetic markers can be used in isolation or in combination with other factors to provide a prognostic tool for influenza infection. Other factors can include patient age, patient gender, patient immune status, patient ethnicity, patient immune status, patient lifestyle factors (e.g. alcohol use, drug use, tobacco use), and/or presence of noninfectious disease (e.g. diabetes, allergy, emphysema, cancer, heart disease).
  • noninfectious disease e.g. diabetes, allergy, emphysema, cancer, heart disease.
  • viral genetic factors can be utilized as part of the prognostic tool or method.
  • patient genetic markers other than those noted above can be used as part of such a prognostic tool or method.
  • markers or factors can be utilized in a multivariate analysis that incorporates PDE3A mutations.
  • Such a multivariate analysis form at least part of a prognostic and/or clinical decision algorithm useful to a clinician in determining a course of treatment for an individual having influenza.
  • polynucleotide amplification technologies such as PCR, reverse transcription PCR, real time PCR, and endpoint PCR.
  • linearization methods such as PCR, reverse transcription PCR, real time PCR, and endpoint PCR.
  • linearization methods such as PCR, reverse transcription PCR, real time PCR, and endpoint PCR.
  • linearization methods such as PCR, reverse transcription PCR, real time PCR, and endpoint PCR.
  • polynucleotide amplification methods can be utilized.
  • the genetic composition of a patient sample can be determined directly (i.e. without amplification).
  • gene products e.g. proteins
  • gene products can be identified by expression level, for example by quantitation relative to a reference protein.
  • specific mutations of a protein gene product can be identified, for example through the use of specific monoclonal antibodies, protein/peptide sequence analysis, production of characteristic fragmentation products on proteolysis, etc.
  • Such methods can be performed manually, using an automated instrument, or in a semi- automated manner. Such methods can provide quantitative, semi-quantitative, or non- quantitative results (e.g. utilizing suitable signal or quantitative cutoff values).
  • the method can be performed in a multiplex fashion (for example using individual indicator fluorophores with non-overlapping emission spectra, a printed microarray, a fluid bead array, or other means of encoding individual results), which permits characterization of two or more mutations from a single sample simultaneously.
  • results are obtained in a single-plex (e.g. monoplex) manner.
  • PDE3A mutations can be identified using an immunological method.
  • specific antibodies can be used to identify the presence of specific mutations using immunoassay techniques (for example immunofluorescence, enzyme immunoassay, or flow immunoassays).
  • an immunoassay can utilize capture of PDE3A from a sample using a specific antibody and subsequent characterization of the capture protein, for example by an activity assay and/or mass spectrometry.
  • primers and probes designed to hybridize to all or part of the PDE3A gene, regulatory region, or regulatory factor.
  • Such primers and probes can include various tags or detection moieties, such as fluorescent dyes, radioactive tags, mass tags, quenching groups, haptenic groups, and so on.
  • Such primers and/or probes can include non- naturally occurring bases and/or sugar-phosphate backbone components in order to optimize binding energy and/or stability.
  • primers and/or probes can include deliberate base mismatches, which can improve specificity.
  • a primer and/or probe sequence can be complementary to a PDE3A sequence from which one or more introns has(have) been excised.
  • compositions and methods of the inventive concept can be applied to any suitable patient sample.
  • suitable samples include body fluids such as blood, saliva, urine, mucus, and other body fluids.
  • Suitable sample can also include tissue samples, such as cheek swabs, skin scrapings, shed epithelial cells, hair follicles, samples obtained by lavage and/or tissue biopsy, and slides obtained for histological studies.
  • rs6487131 is located within the predicted binding site of transcription factors CTCF, Rad21 and USF1. The genetic variations in such a transcription factor binding sites can affect the transcription activity of PDE3A.
  • GTEx Genotype-Tissue Expression
  • PDE3A can act as an antiviral host factor in influenza infection and diseases (mutations in which can lead to severe disease on infection with influenza). Inventors also believe that PDE3A can also affect disease severity via other physiological functions.
  • known physiological functions of PDE3A include the regulation of vascular smooth muscle contraction and relaxation in the heart, insulin secretion, lipolysis, oocyte maturation, and platelet aggregation.
  • Another embodiment of the inventive concept is a method of treating influenza, in particular severe influenza.
  • a method can include treating a patient with pro-inflammatory cytokines and/or inducing a patient to express pro-inflammatory cytokines.
  • Suitable proinflammatory cytokines include TNFa, IL- ⁇ , IL-6, and IL-32.
  • pro -inflammatory cytokines can be supplied as prodrugs and/or in modified form.
  • suitable proinflammatory cytokines can be provided as conjugates (for example, polyethylene glycol conjugates).
  • an individual with influenza can be treated with compounds that increase production and/or release of pro-inflammatory cytokines.
  • Another embodiment of the inventive concept is a method of treating influenza by modulating the expression of PDE3A and/or the use of PDE3A agonists.
  • a person with influenza can be treated using a PDE3A-inducing compound.
  • a person with influenza can be treated with a compound that acts as a PDE3A agonist to provide an effect similar to that of increased PDE3A production and/or release.
  • induction can provide levels of PDE3A activity that are at least equivalent to that of a normal individual in a person with a PDE3A mutation that affects expression and/or activity of PDE3A gene product.
  • such therapy can provide PDE3A activity that exceeds that of a normal individual by 10%, 50%, 100% or more in normal individuals and/or individuals carrying PDE3A mutations that impact expression and/or activity of the PDE3A gene product.
  • Genotyping of patients with A(HlNl)pdm09 infection The first cohort of patients was genotyped using Genome -Wide Human SNP Array 6.0TM (Affymetrix Inc.). The SNPs rs7314545, rs6487131 and rs6487132 were genotyped using the MassARRAY SystemTM
  • Genomic DNA for genotyping was obtained from archived blood specimens or respiratory tract specimens.
  • Genotype data for the general population of Han Chinese Subjects were recruited with written informed consent from the University Hospital, Macau University of Science and Technology (MUST), Macau between 2014 and 2015. Only Chinese subjects were included. All samples were genotyped using Human Omni ZhongHua-8 BeadChipsTM (Illumina) following the manufacturer's instructions. The SNPs included rs6487132 and rs6487131. However, rs7314545 was not included in the chip. Additional genotype data for Han Chinese were also extracted from the data of Han Chinese in Beijing (CHB) and Southern Han Chinese (CHS) in the 1000 Genomes Project Phase 3.
  • CHB Han Chinese in Beijing
  • CHS Southern Han Chinese
  • Viruses and cell lines All virus strains used in this study were clinical strains, and included influenza virus subtypes A(HlNl)pdm09 (A/Hong Kong/415742/2009 [H1N1-2009] and A/Hong Kong/402467/2014 [HlNl-2014] ), A(H3N2) (A/Hong Kong/447572/2011 [H3N2- 2011]), A(H5N1) (A/Vietnam/ 1194/2004 [H5N1-2004]) and A(H7N9) (A/Anhui/1/2013 [H7N9- 2013-AH1] and A/Zhejiang/DTID-ZJU01/2013 [H7N9-2013-ZJ1]) [12, 21]. These viruses were propagated in Madin Darby canine kidney (mdck) cells. Human alveolar epithelial cell line A549 and human bronchial epithelial cell line Calu-3 were used for in vitro experiments
  • A549 cells were seeded onto 96-well plates with Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.025 M of HEPES buffer, 100 units/ml of penicillin G, 100 ⁇ g/ml of Streptomycin, 20 ⁇ g/ml of gentamicin and 20 units/ml of nystatin (all reagents from Thermo Fisher Scientific, MA, USA). The culture plates were incubated at 37° C and 5% C0 2 .
  • DMEM Dulbecco's Modified Eagle's medium
  • FBS fetal bovine serum
  • Calu-3 cells the conditions were the same as for A549 cells except that the culture medium before infection contains DMEM/Nutrient Mixture F-12 (DMEM/F12) instead of DMEM, and Calu3 cells were inoculated with the A(HlNl)pdm09 virus with 0.5 ⁇ ⁇ TPCK- treated trypsin instead of 1 ⁇ g/ml.
  • DMEM/F12 DMEM/Nutrient Mixture F-12
  • A549 cells were transfected with siRNA targeting PDE3A gene (PDE3A siRNA) (siRNA ID S 10184, Thermo Fisher Scientific, MA, USA), PDE3B gene (PDE3B siRNA) )(siRNA ID S 10188, Thermo Fisher Scientific, MA, USA) or non-targeting control siRNA (SilencerTM Select Negative Control No. 2 siRNA, Thermo Fisher Scientific, MA, USA).
  • RNAiMAX transfection reagent (Thermo Fisher Scientific, MA, USA) according to manufacturer's instructions. The knockdown efficiency was determined by comparing the PDE3A mRNA and protein expression of PDE3A-siRNA transfected cells and those of control-siRNA transfected cells. PDE3A mRNA expression was determined using RT- qPCR as described above. PDE3A protein expression was determined using Western blot as we described previously with modifications [12]. Rabbit anti-PDE3A antibody (Abeam, United Kingdom) was used as the primary antibody.
  • Multicycle growth assay in A549 cells A549 cells transfected with PDE3A siRNA or non-targeting control siRNA were inoculated with different influenza virus strains at an MOI of 0.01. Viral titer in the culture supernatant at predetermined time points post-infection was determined by plaque assay as we described previously [12].
  • Influenza A nucleoprotein expression of A549 infected with H IN 1-2009 A549 cells transfected with PDE3A siRNA or non-targeting control siRNA were inoculated with influenza virus strain H1N1-2009 at an MOI of 10. Influenza A nucleoprotein expression was determined by Western blotting analysis.
  • RNA expression of cytokines and chemokines were determined using RT-qPCR as described previously [22]. RNA extraction and RT-qPCR were performed as described for PDE3A mRNA expression, except that specific primers for the cytokines and chemokines were used (see Table 1).
  • PDE3A gene variants were significantly associated with disease severity.
  • the first cohort of 84 patients consisted of 42 severe and 42 mild cases of A(HlNl)pdm09 infection who were matched for age, gender and underlying conditions, and the SNPs of these severe and mild cases were compared. The comparison of the demographics and underlying risk conditions between severe and mild cases in this first cohort has been described previously [15] . Although none of the SNPs reached the cutoff for genome- wide significant result of P ⁇ 5xl0-8, the PDE3A gene SNPs rs7314545, rs6487131, and rs6487132 had odds ratio >15 and allelic association with P ⁇ 0.001 (see FIG. 2).
  • the odds ratio ranged from 3.38-3.96, with allelic association P values between 10 ⁇ 6 to 10 ⁇ 5 (see Table 2).
  • the SNPs rs6487131 and rs6487132 were in complete linkage based on available data in the general Han Chinese population.
  • the SNP rs7314545 was also highly linked to rs6487131/rs6487132.
  • One hundred and two other SNPs related to PDE3A were also screened but the P values were all above 0.001 when comparing between severe and mild cases (see Table 3).
  • 2C shows a comparison of population genetic risks among Chinese and Caucasian populations using the genetic markers rs6487131, rs2070788 and rsl l30866 of the PDE3A gene.
  • the Southern Chinese population in Macau showed a significantly higher genetic risk of having severe syndromes after H1N1 pdm09 infection when compared with American and European populations (P ⁇ 0.001, Kruskal- Wallis test; P ⁇ 0.001, Dunn's pairwise comparison) (see FIG. 2C, upper panel).
  • P 0.067, Kruskal- Wallis test
  • PDE3A is a cyclic nucleotide phosphodiesterase which regulates the intracellular levels of the cellular second messenger cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). There is no previously known association between PDE3A and pathogenesis of viral infections.
  • cAMP cyclic adenosine monophosphate
  • cGMP cyclic guanosine monophosphate
  • A(HlNl)pdm09 virus infection upregulated PDE3A mRNA expression in human lung epithelial cell lines To determine the effect of A(HlNl)pdm09 virus infection on PDE3A mRNA expression, alveolar epithelial cell A549 and bronchial epithelial cell Calu-3 were infected with HlNl-2009, and expression of PDE3A mRNA in the cell lysates was determined. PDE3A mRNA expression was significantly higher in HlNl-2009-infected A549 and Calu-3 cells than those of non-infected cells (see FIG. 3). Hence, HlNl-2009 infection induces PDE3A expression in human airway epithelial cells.
  • PDE3A knockdown enhanced viral replication of A(HlNl)pdm09, A(H5N1), A(H7N9), but reduced viral replication for A(H3N2) virus:
  • virus replication between A549 cells with knockdown of PDE3A using PDE3A- specific siRNA was compared with A549 cells transfected with control siRNA.
  • PDE3A knockdown was confirmed by both RT-qPCR and by Western Blot analysis (see FIG. 4).
  • PDE3A siRNA used in our experiments did not significantly affect the expression of other phosphodiesterases tested, specifically PDE3B, PDE4A, PDE4B, PDE4D and PDE8A (see Table 6).
  • the viral titers in the PDE3A knockdown cells were also significantly higher for H1N1- 2014, H5N1-2004, H7N9-2013-AH1 and H7N9-2013-ZJ1 at 48 hours post-infection.
  • influenza A nucleoprotein expression in A549 cells after HlNl-2009 infection was determined. At 4 hours post-infection, influenza nucleoprotein expression was significantly enhanced in PDE3A-knockdown A549 cells than that of control siRNA-transfected A549 cells (see FIG. 5C).
  • PDE3A knockdown and cytokine expression during influenza virus infection The effect of PDE3A knockdown on cytokine and chemokine expression after HlNl-2009 infection of A549 cells was determined by RT-qPCR. PDE3A knockdown led to significant increased mRNA expression of IL-6 and IL-32 in the cell lysate ( Figure 4). However, there was no significant difference in the mRNA expression of IL10, TNF-a, IP- 10, and IFN- D between PDE3A knockdown A549 cells and control cells (data not shown).
  • Hung IF To KK, Lee CK, et al. Hyperimmune IV immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 influenza A(H1N1) infection. Chest 2013; 144:464-73.
  • Ahmad F Degerman E, Manganiello VC. Cyclic nucleotide phosphodiesterase 3 signaling complexes. Horm Metab Res 2012; 44:776-85.
  • Cilostazol protects mice against endotoxin shock and attenuates LPS -induced cytokine expression in RAW 264.7 macrophages via MAPK inhibition and NF-kappaB inactivation: not involved in cAMP mechanisms.
  • Cilostazol protects mice against endotoxin shock and attenuates LPS -induced cytokine expression in RAW 264.7 macrophages via MAPK inhibition and NF-kappaB inactivation: not involved in cAMP mechanisms.
  • Cilostazol ameliorates systemic insulin resistance in diabetic db/db mice by suppressing chronic inflammation in adipose tissue via modulation of both adipocyte and macrophage functions. Eur J Pharmacol 2013; 707: 120-9.
  • Boda H Uchida H, Takaiso N, et al. A PDE3A mutation in familial hypertension and brachydactyly syndrome. J Hum Genet 2016; 10.1038/jhg.2016.32.
  • Tetherin sensitivity of influenza A viruses is strain specific: Role of hemagglutinin and neuraminidase. J Virol 2015; 10.1128/JVI.00615-15.

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Abstract

L'invention concerne des compositions et des procédés qui servent à déterminer le pronostic de la gravité d'une infection grippale sur la base de l'état de PDE3A d'un individu. Une fonction de PDE3A altérée est associée à des symptômes graves si une grippe est contractée. Des mutations de SNP spécifiques sont identifiées, lesquelles sont associées à une fonction de PDE3A altérée, et des amorces et des kits qui permettent l'identification de ces SNP sont fournis. De telles informations peuvent également être combinées à d'autres données de patient.
PCT/US2017/040368 2016-07-01 2017-06-30 Compositions et procédés servant au pronostic d'une infection grippale WO2018006014A1 (fr)

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WO2004071377A2 (fr) * 2003-02-17 2004-08-26 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques relatives a des etats pathologiques lies a la photodiesterase 3a (pde3a)
WO2008157518A1 (fr) * 2007-06-15 2008-12-24 Drake Richard R Biomarqueurs d'une réponse à un vaccin de la grippe
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